NUCLEIC ACID MODIFICATION WITH SPECIFIC ENDONUCLEASES

长期目标是表征有用的核酸酶和其他 来自海洋细菌属交替单胞菌的胞外酶。 的 该项目的健康相关性在于使用这些酶， 与基因工程相关的核酸操作， 核酸酶已被证明可用于克隆， 在大肠杆菌中的表达含有胰岛素等医学上重要的蛋白质的大肠杆菌 和干扰素。 核酸酶存在于至少两个分子中， 和动力学上不同的形式，并拥有活动， 单链DNA、双链DNA中的共价损伤和其他畸变 和线性双链体DNA的末端，进一步表征为 关于催化行为对许多因素的依赖性， 环境变量。 动力学和反应机理研究 包括中间体和最终产品的表征 消化的DNA，阐明的方向性和持续性， 对线性双链体DNA和单链DNA的活性， 2 ′，3 ′-双脱氧末端核苷酸和带有 当这样的α-磷酸上的硫原子对核酸外切酶活性的影响时， 基团存在于末端， 切割的环状双链体DNA转化为线性双链体DNA， 切割位点在含有预先存在的 链断裂到初始断裂的位点。 一个已知的模型 产生带有单链末端的可连接片段， 将测试线性双链DNA的部分降解，这是预期的 从而得到一种检测回文序列的通用方法， 双链DNA 非超螺旋闭合环的裂解动力学 DNA与致癌物质苯并[a]芘的代谢物反应后， 测定 一种核酸酶显然可以来源于 其他通过蛋白水解，这是要确认和观察的中间体 考察 确定核酸酶是否以潜在的方式切割 超螺旋DNA中的十字形结构，这已被证明是 将进行其它单链特异性核酸酶的情况。 尝试 克隆核酸酶的基因将首先使用 本实验室开发的简单方法， 周质或细胞外核酸酶。 A的初步特征 培养物上清液中具有5 ′-核苷酸酶活性的磷酸酶将 进行。

The long-term objectives are to characterize useful nucleases and other extracellular enzymes from the marine bacterial genus Alteromonas. The health relatedness of the project lies in the use of these enzymes in genetic engineering-related manipulations of nucleic acids, for which the nucleases have already proven useful in connection with the cloning and expression in E. coli of such medically significant proteins as insulins and interferons. The nucleases, which exist in at least two molecularly and kinetically distinct forms and possess activities against single-stranded DNA, covalent lesions and other distortions in duplex DNA and the termini of linear duplex DNA, are to be further characterized with respect to the dependence of catalytic behavior on a number of environmental variables. Kinetic and reaction mechanisms studies to be done include characterization of the intermediate and final products of digestion of DNA, the elucidation of the directionality and processivity of the activities on linear duplex DNA and single stranded DNAs, the effects of 2',3'-dideoxy terminal nucleotides and of nucleotide analogs bearing a sulfur atom on the Alpha-phosphate on the exonuclease activity when such groups are present at the termini, measurement of the kinetics of conversion of nicked circular to linear duplex DNA and the determination of the location of the site of cleavage in DNA containing a pre-existing strand break to the site of the initial break. A model for the known generation of ligatable fragments bearing single-stranded termini after partial degradation of linear duplex DNAs will be tested, which is expected to lead to a general method for the detection of palindromic sequences in duplex DNAs. The kinetics of cleavage in nonsupercoiled closed circular DNA reacted with a metabolite of the carcinogen benzo[a]pyrene will be determined. One species of the nuclease can apparently be derived from the other by proteolysis and this is to be confirmed and observed intermediates examined. Determination of whether the nuclease cleaves at potential cruciform structures in supercoiled DNA for which this has been shown to be the case for other single-strand-specific nucleases will be done. Attempts to clone the gene(s) for the nuclease will be made, using as first approaches simple methods developed in this laboratory to assay for periplasmic or extracellular nucleases. Preliminary characterization of a phosphatase in culture supernatants that has 5'-nucleotidase activity will be undertaken.